Cephalosporin C (hereinafter, referred to as “CPC”) is one of β-lactam family antibiotics produced by filamentous fungi such as Acremonium chrysogenum. CPC shows antibiotic activity against Gram-negative bacteria by hindering cell wall synthesis, but it is not active enough against the growth of Gram-negative bacteria. Accordingly, CPC has been mainly used for preparing intermediates for the production of semi-synthetic cephalosporin antibiotics. In particular, 7-aminoacephalosporanic acid (hereinafter, referred to as “7-ACA”) prepared by removing the D-α-aminoadipoyl side chain from CPC has been used for the production of most semi-synthetic cephalosporin antibiotics that account over 40% share of the world antibiotics market.
There are two known methods for preparing 7-ACA from CPC, chemical and enzymatic methods. The chemical method of synthesizing 7-ACA from CPC uses silyl protecting groups for both amine and carboxyl groups and gives a yield of over 90%. However, this method is complicated, uneconomical and environmentally unfavorable. Therefore, the chemical method has been replaced by an enzymatic method for preparing 7-ACA which is regarded as an environmentally acceptable method.
A two-step enzymatic method widely used commercially at present comprises the two steps of converting CPC into glutaryl-7-aminocephalosporanic acid (hereinafter, referred to as “GL-7-ACA”) by D-amino acid oxidase (hereinafter, referred to as “DAO”) (the first step) and GL -7-ACA into 7-ACA by GL-7-ACA acylase (the second step) (see FIG. 1). However, this method gives a lower yield of 7-ACA than the chemical method, due to a large quantity of by-products generated by the reaction of hydrogen peroxide produced in the first step with the DAO substrate or the reaction product. Therefore, there has been a need to develop an efficient one-step enzymatic method for directly converting CPC into 7-ACA using a modified CPC acylase which is capable of breaking an amide linkage at the 7th position of CPC and removing the aminoadipoyl side chain.
CPC acylase (also called together with CPC amidase or CPC amidohydrolase) active toward CPC has been found in several microorganisms, such as Pseudomonas sp., Bacillus megaterium, Aeromonas sp., Arthrobacter viscous etc., and some CPC acylase genes have been cloned and sequenced: Pseudomonas sp. SE83 derived acyII gene (Matsuda et al., J. Bacteriol. 169: 5821-5829, 1987); Pseudomonas sp. N176 derived CPC acylase gene (U.S. Pat. No. 5,192,678); Pseudomonas sp. V22 derived CPC acylase gene (Aramori et al., J. Ferment. Bioeng. 72: 232-243, 1991); Pseudomonas vesicularis B965 derived CPC amidohydrolase gene (U.S. Pat. No. 6,297,032); Bacillus megaterium derived CPC amidase gene (U.S. Pat. No. 5,229,274); and Pseudomonas sp. 130 derived CPC acylase gene (Li et al., Eur. J. Biochem. 262: 713-719, 1999). However, these CPC acylases are not hydrolytically active enough to cleave the amide linkage at the 7th position of CPC, and thus, it is not suitable for a one-step enzymatic process for preparing 7-ACA from CPC.
Several genetic engineering studies have been attempted to increase the enzyme activity of CPC acylase toward CPC. For example, Fujisawa Pharmaceutical Co. (Japan) developed a CPC acylase mutant derived from Pseudomonas sp. N176 which shows about 2.5-fold higher specific activity toward CPC than that of a wild-type (U.S. Pat. No. 5,804,429; U.S. Pat. No. 5,336,613, Japanese Patent Publication No. 1995-222587; Japanese Patent Publication No. 1996-098686; and Japanese Patent Publication No. 1996-205864). However, such a mutant still has insufficient specific activity toward CPC to directly produce 7-ACA from CPC and exhibits end-product inhibition by 7-ACA. Therefore, it can't be used practically in the direct conversion of CPC to 7-ACA.
For the purpose of developing a CPC acylase mutant applicable to a one-step enzymatic method for preparing 7-ACA, the tertiary structure of GL-7-ACA acylase has been investigated by the present inventors, who have identified for the first time the tertiary structures of apoenzyme (Kim, et al., Structure 8: 1059-1068, 2000) and CAD-GL-7-ACA complex (Kim, et al., Chem. Biol. 8: 1253-1264, 2001; Kim, et al., J. Biol. Chem. 276: 48376-48381, 2001) associated with GL-7-ACA acylase derived from Pseudomonas sp. KAC-1 (Kim, et al., Biotech. Lett. 23: 1067-1071, 2001; hereinafter, referred to as “CAD”) (see FIG. 2). The structures of the two CAD binary complexes suggest that the most extensive interactions between the GL-7-ACA acylase and a substrate took place in the glutaryl moiety of GL-7-ACA. Therefore, it is suggested that the hydrophilic and hydrophobic interactions between the side-chain of substrate and its binding pocket are the dominating factors in recognizing the substrate in GL-7-ACA acylase. When the chemical structures of CPC having extremely lower substrate-binding affinity and GL-7-ACA are compared, their β-lactam structures are the same, but there are some differences in the side chain (see FIG. 3). Namely, unlike the side chain of GL-7-ACA which is composed of glutaric acid, that of CPC is a D-α-aminoadipic acid. Therefore, the modeling of the structure of CAD-CPC complex suggests that the glutaric acid side chain of GL-7-ACA and the binding-related key residues of CAD are sterically crowded with respect to the carboxyl and D-form amino groups at the terminal end of D-α-aminoadipic acid side chain (see FIG. 4). Thus, if an enough space for accommodating the CPC side chain (that contains a carbon backbone greater than that of the GL-7-ACA side chain and the D-amino group) can be secured in the substrate binding site of CAD, it is considered that the specific activity of GL-7-ACA for CPC can be increased. Accordingly, the structural analysis about the “enzyme-substrate” complex of CAD may provide important information for developing a CPC acylase mutant having an increased specific activity to CPC by introducing the mutations at the active site of GL-7-ACA acylase derived from Pseudomonas sp. that show relatively low substrate-binding affinity.
The present inventors have therefore endeavored to develop a CPC acylase mutant having improved reactivity to CPC which can be used in a one-step enzymatic method for preparing 7-ACA from CPC.